Project Summary: a) Pre-treatment Tumor pO2 and metabolic profile in tumor bearing mice to predict efficacy of radiotherapy and hypoxia activated pro-drugs: Solid tumors have vasculature characterized by poor architecture and function resulting in leaky vessels, poor delivery of oxygen and nutrients resulting in hypoxia and low pH. These microenvironmental features such as hypoxia result in resistance to radiotherapy and chemotherapy with anti=proliferative drugs. Hypoxia activated prodrugs can be used to treat tumors with chronic hypoxia while radiation and antiproliferative agents can be used in tumors with higher oxygen levels. Here we have studied three pancreatic tumor xenografts SU86.86, MiaPaca-2, Hs766t for their physiologic (pO2) and metabolic (glycolysis) status using EPRI and metabolic MRI. We find that the pO2 status follows Su86/86MiaPaca-2Hs766t. Similarly, the glycolysis efficiency, as monitored using metabolic MRI using the conversion of hyperpolarized pyruvate to lactate showed that the trend to be Hs766tMiaPaca-2SU86.86. Thus this study established for usefulness of both these imaging biomarkers to categorize tumors on the basis of physiologic and metabolic basis. With information provided by these biomarkers, we set up survival studies of separate groups of mice bearing Su86.86, MIaPAca-2, and Hs766t untreated (control), X-ray treated (3-fractions of 5Gy each), the hypoxia activated prodrug TH-302, or gemcitabine. The studies show that radiotherapy efficacy followed Su86.86MIapaca-2Hs766t similar to the trend of oxygen status and opposite to the trend of glycolysis. Similarly the efficacy of the hypoxia activated TH-302 followed the trend Hs766tMiaPaca-2SU86.86. These studies support the strength of imaging biomarkers in predicting treatment response as well as in aiding treatment combinations. b) Evaluating the requirement of oxygen in photoimmunotherapy: Photoimmunotherapy using an antibody specific to EGFR linked to a near infrared light absorbing chromophore (IR700) has shown remarkable efficacy in many pre-clinical models and is on the verge of entering clinical phase. This study was designed to understand the mechanistic aspects of the actionof PIT using cetuximab-IR700 immunoconjugate in chemical, cellular and in vivo models. In chemical studies, it was found that the Cetuximab-IR700 generates reactive oxygen species such as singlet oxygen copiously. IN cellular studies, oxygen was found to be essential for the cytotoxicity of PIT mediated by Cetuximab-IR700. Similarly in in vivo studies, when oxygen supply was limited to tumor temporarily by applying a tourniquet during PIT, no effect of treatment was noticed. These data establish the critical role molecular oxygen plays in the efficacy of PIT and helps manage the treatment protocol clinically by exploring ways to improve oxygen during PIT. Since the tumor control by PIT is essentially by rapid necrosis, we examined imaging biomarkers which can report in vivo nectrotic processes. With cell membrane destruction and leakage of mitochondrial enzymes in the TCA cycle, we examined if the conversion of fumarate to malate can be imaged with hyperpolarized MRI. We were able to successfully polarize 13C labeled fumarate so that there is sufficient sensitivity to conduct 13C MRSI. In treated tumors, we were able to detect the conversion of fumarate to malate suggesting rapid onset of necrosis following PIT